This invention generally relates to humanized anti-CD40 antibodies for diagnostic and therapeutic use. More specifically, humanized anti-CD40 antibodies and methods of use for the treatment of various diseases or disorders characterized by cells expressing CD40 are disclosed. Pharmaceutical compositions and articles of manufacture such as kits comprising the humanized anti-CD40 antibody are also disclosed.
CD40 is a type I integral membrane glycoprotein and a member of the tumor necrosis factor (TNF) receptor superfamily. CD40 is expressed on a variety of cell types including normal and neoplastic B cells, interdigitating cells, basal epithelial cells and carcinomas. It is also present on monocytes, macrophages, some endothelial cells, and follicular dendritic cells. CD40 is expressed early in B cell ontogeny, appearing on B cell precursors subsequent to the appearance of CD10 and CD19, but prior to expression of CD21, CD23, CD24, and appearance of surface immunoglobulin M (sIgM) (Uckun et al., 1990, Blood 15:2449). Although early reports indicated that CD40 was lost upon terminal differentiation of B cells into plasma cells, CD40 has been detected on tonsil and bone marrow-derived plasma cells (Pellat-Decounynck et al., 1994, Blood 84:2597).
The interaction of CD40 with its ligand and counter-receptor, CD40L (also referred to as CD154, gp39, and TRAP), induces both humoral and cell-mediated immune responses. CD40L is a transmembrane protein expressed predominantly on activated lymphocytes.CD4+ T cells. Like other proteins in the TNF family, the structure of CD40L is that of a noncovalent trimer. CD40-mediated signaling appears to be required for B cell proliferation, immunoglobulin (Ig) isotype switching, germinal center formulation, and memory B cell commitment in response to T cell-dependent antigen. CD40 binding of CD40L results in CD40 multimerization, the generation of activation signals for antigen presenting cells such as dendritic cells, monocytes, and B cells, and the generation of growth and differentiation signals for cytokine-activated fibroblasts and epithelial cells. While the signaling pathways through which CD40 molecules function in cell differentiation have not been completely elucidated, CD40 signals are transduced from the multimerized receptor via recruitment of a series of TNF receptor associated factors (“TRAFs”) (Kehry, 1996, J. Immumol. 156:2345-2348). Subsets of TRAFs interact differentially with TNF receptor family members, including CD40, providing stimuli to a wide variety of downstream pathways. TRAF1 and TRAF2 are implicated in the modulation of apoptosis (Speiser et al., 1997, J. Exp. Med. 185:1777-1783; Yeh et al., 1997, Immunity 7:715-725). TRAFs 2, 5, and 6 participate in proliferation and activation events. In normal B cells, binding of CD40 to CD40L recruits TRAF2 and TRAF3 to the receptor complex and induces down regulation of other TRAF's (Kuhne et al., 1997, J. Exp. Med. 186:337-342).
Apoptosis and CD40-mediated signaling are closely linked during B cell development and differentiation. A primary function of apoptosis in B cells is the clonal deletion of immature B cells, which is thought to result from extensive cross-linking of surface Ig in immature B cells. The fate of mature B cells is also modulated by a combination of signaling via surface Ig and signals derived form activated T cells, presumably mediated by CD40L molecules. A combination of signals from surface Ig and CD40 can override the apoptotic pathway and maintain germinal center B cell survival. This rescue from apoptosis in germinal centers is critical for the development of affinity antibody-producing memory B cells.
In both T and B cell malignancies, antitumor effects (growth arrest with or without apoptosis) often result when malignant cells are exposed to stimuli that lead to activation of normal lymphocytes. This activation-induced growth arrest has been observed with signals through either antigen receptors or costimulatory receptors (Ashwell et al., 1987, Science 237:61; Bridges et al., 1987, J. Immumol. 139:4242; Page and Defranco, 1988 J. Immunol. 140:3717; and Beckwith et al., 1990, J. Natl. Cancer Inst. 82:501). CD40 stimulation by either anti-CD40 antibody or soluble CD40L directly inhibits B cell lymphoma growth (Funakoshi et al., 1994, Blood 83:2787-2784).
Several murine monoclonal antibodies (mAbs) directed against CD40 have been described (Katira et al. 1995, “CD40 Workshop Panel Report”; In: Leukocyte Typing V, Schlossman et al., (eds) 1995, 1:547-550). For example, two mAbs, CD40.7 (M2) and CD40.8 (M3), were shown to inhibit the binding of CD40 to CD40L (Fanslow et al., 1995, In: Leukocyte Typing V, Schlossman et al., (eds) 1995, 1:555-556). CD40 stimulation by mAbs M2 and M3 inhibited growth of several human B-cell lymphomas and induced regression of established tumors in vivo (Funakoshi et al., 1994, Blood 83:2787-2794; Funakoshi et al., 1996, J. Immunol. 19:93-101). U.S. Pat. No. 5,182,368 discloses an anti-CD40 murine mAb, G28-5, which can augment B cell proliferation. A single chain immunotoxin based the single-chain Fv region of G28-5 selectively killed human CD40-expressing hematologic malignant cell lines in vitro (Francisco et al., 1997, J. Biol. Chem. 39:24165-24169). However, G28-5 does not enhance activation of B cells in the presence of CD40L and does not potentiate the binding of CD40 and CD40L. U.S. Pat. No. 6,838,261 (and related U.S. Pat. Nos. 6,946,129 and 6,843,989) describes a class of variant forms of the anti-CD40 murine mAb, S2C6, and its use in the treatment of various disorders, including cancer and immunological and inflammatory diseases. In addition to enhancing CD40L-mediated stimulation, an anti-CD40 antibody described in U.S. Pat. No. 6,838,261 showed enhancement of the interaction between CD40 and CD40L, and in vivo anti-neoplastic activity. Although S2C6 by itself will stimulate B cell proliferation in a manner similar to G28-5, S2C6 is distinguished from G28-5 by its ability to increase CD40L binding and the subsequent magnitude of the CD40L-mediated activation signal.
Other murine anti-CD40 mAbs, e.g., described in International Publication Number WO 95/17202, bind CD40 and show efficacy in the treatment and prevention of disease characterized by neoplastic cells expressing CD40. Although murine anti-CD40 antibodies have potential applicability as therapeutic agents in the treatment of CD40-related diseases in humans, their immunogenicity presents the possibility of a neutralizing antibody response, e.g., a human anti-mouse antibody (HAMA) response which would limit their value.
Thus, there is a need for humanized anti-CD40 antibodies that specifically bind defined CD40 epitopes and which show the antigen binding specificity, affinity, and other desired functional characteristics of the analogous nonhuman anti-CD40 antibody.